Stable hemoglobin reference solution

ABSTRACT

A stable reference solution for calibrating and monitoring blood gas instrumentation is disclosed. The solution comprises an aqueous mixture containing a hemoglobin solution derived from a mammalian source which comprises at least about 95% reduced hemoglobin. The solution additionally contains a bicarbonate buffer and a metal catalyzed methemoglobin reducing system and an organic buffer. To provide a control element having a variety of properties similar to fresh, whole, human blood, the reference solution is stored in a sealed ampule under an inert atmosphere containing CO 2  until just prior to use. The ampule is subsequently opened and the solution is equilibrated with a gas mixture having components which provide gasses in a physiological range to the solution. The solution is characterized by exhibiting properties similar to fresh blood subsequent to equilibration and an extended storage life prior to equilibration.

This application is a continuation-in-part of Ser. No. 07/328,622 filedMar. 27, 1989, now U.S. Pat. No. 5,045,529, issued Sep. 3, 1991.

BACKGROUND OF THE INVENTION

In practice, the use of analytical equipment for measuring variousparameters of blood requires utilization of control solutions whichexhibit properties as close as possible to those observed in freshnormal human blood. By measuring known parameters of control solutions,the instrumentation can be monitored and calibrated to allow highlyaccurate measurements of patient blood samples.

One approach to monitoring the reliability and accuracy of instrumentsthat measure partial pressures of CO₂ and O₂ in blood is with the use ofsamples of fresh human blood which has been tonometered with gasmixtures having known amounts of CO₂, O₂ and N₂. Such a process isdescribed by Burnett in Clinical Chemistry, 27(10):1761 (1981).

When the blood has been properly tonometered, the sample will haveprecise and fixed partial pressures of CO₂ and O₂. These preparedsamples can then be introduced into the analytical instrument and pCO₂and pO₂ values determined. Since the sample is similar to a patientblood specimen, but with known pCO₂ and pO₂ values, the instrument canbe considered reliable for measuring unknown patient samples if the testvalues of the tonometered blood equal the theoretical values based onthe gas mixture used for tonometry.

Although the use of tonometered blood is considered to be satisfactoryfor monitoring blood gas instrumentation, the approach has a number ofdrawbacks which limit its use in all but a very small percentage oflaboratories.

For example, blood samples derived from human sources are susceptible toinfectious agents, including hepatitis virus and HIV which can poseserious health hazards to laboratory personnel who must perform thetonometry and testing of the sample. In addition, the instrumentscommonly used to measure pCO₂ and pO₂ also measure blood pH. Since thetonometered blood does not have a known pH value, the sample cannot beused for monitoring the pH measurements and a separate pH controlstandard must be used.

Similarly, many laboratories that perform measurements of pCO₂, pO₂ andpH on blood samples also measure total hemoglobin and hemoglobinfractions on a CO-Oximeter that is located near the blood gasinstrument. However, since the blood used for tonometry is acquired fromrandom patient samples, the tonometered sample has no known hemoglobinvalue, and therefore it is not useful for monitoring the CO-Oximeter.Consequently, a separate control standard is required for thisinstrument also.

Finally, the entire procedure for properly preparing tonometered bloodsamples requires disciplined techniques and many laboratories lacktrained personnel as well as the time to prepare the samples.

Because of these disadvantages, most laboratories use control standardswhich mimic human blood but have properties quite different than freshblood. For example, buffered aqueous solutions which have beentonometered with CO₂ and O₂ are often used. These materials are assayedfor predetermined values for pH, pCO₂ and pO₂. However, in composition,physical properties and chemical properties they differ greatly fromwhole blood.

Other control standards comprise buffered suspensions of modified humanred blood cells or hemoglobin solutions prepared from lysed red bloodcells. These materials have some properties which more closelyapproximate actual blood than do the aqueous based controls, but theirpO₂ buffering action and inability to provide the O₂ saturationcharacteristics of fresh blood, cause these materials to perform morelike aqueous solutions than tonometered fresh blood. Furthermore, sincethey are prepared from human blood, the health risk to technicians isnot eliminated.

In summary, the commercial blood gas controls which are used instead oftonometered fresh human blood are generally considered a compromisebetween convenience, economy and the ideal control standard.

A need exists for a reference solution which is not susceptible toinfection, which can be packaged to eliminate the sample collection andpreparation steps necessary when blood samples are used and is stable inthe packaged form. Further, the solution should be capable of beingtonometered in the same manner as fresh human blood to provide a controlstandard that has O₂ saturation characteristics and other propertiessimilar to fresh human blood, but unlike the blood samples, will havepredetermined pH and hemoglobin values for monitoring the instrumentperformance in the measurement of these parameters as well as pO₂ andpCO₂.

Although some of the commercial blood based materials can be used asprepackaged fluids for tonometry, the inability of these materials to bemanufactured and stored without the oxidation of a significant percentof the hemoglobin to methemoglobin (which does not bind with oxygen),causes the solution to lose the oxygen saturation properties of freshblood, and therefore makes the material unsuitable as a substitute forblood as a tonometry solution.

Attempts to provide a system for reducing methemoglobin content in ablood-based material have been the subject of a variety of scientificstudies. For example, in U.S. Pat. No. 4,485,174 to Chiang et al., a"methemoglobin reductase" enzyme system is described as a means formaintaining a low methemoglobin level. This system, however, hasdemonstrated only limited usefulness, since the supply of methemoglobinreducing reagents can be exhausted when the hemoglobin solution isstored under an oxygen containing atmosphere.

Other blood-gas control solutions and methods have been described inU.S. Pat. No. 3,859,049; U.S. Pat. No. 3,973,913; U.S. Pat. No.4,001,142; and U.S. Pat. No. 4,469,792. Additionally, a blood-gascontrol solution and method has been described by Steiner et al.,Clinical Chemistry, 24, 793 (1978). Each of these, however, described acontrol standard having a limited storage life and/or a chemicalformulation which provides physiologically inaccurate values.

Thus, a need still exists for a blood-based reference solution which,after equilibration with an appropriate gas mixture, can be used tomonitor blood-gas analysis equipment. The reference solution shouldpreferably have an extended storage life, provide physiologicallyaccurate blood-gas values, and provide uniform values among a largenumber of samples.

SUMMARY OF THE INVENTION

The present invention pertains to a stable hemoglobin solution which canbe used as a control reference for blood-gas analysis instrumentation aswell as CO-Oximeters. More specifically, the present invention pertainsto an aqueous hemoglobin-containing solution which can be equilibratedwith a variety of gasses generally present in blood to provide asolution having a variety of parameters similar to those parametersnormally found in fresh whole blood. Such an equilibrated solution hasutility as a control in blood-gas analysis and CO-Oximetryinstrumentation.

One embodiment of the invention is an aqueous solution comprising:

a) hemoglobin;

b) a source of bicarbonate ions to stabilize the pH of the solutionfollowing equilibration with a predetermined gas mixture;

c) a source of thio functional groups to maintain a low level ofmethemoglobin;

d) a source of metal ions to catalyze the methemoglobin reductionreaction;

e) at least one organic polyphosphate; and

f) an organic buffer.

To maintain anaerobic conditions, the mixture is stored under an inertatmosphere (preferably nitrogen) containing CO₂. The organicpolyphosphates act to stabilize the hemoglobin molecules in a spatialconfiguration which is not easily amenable to oxidation tomethemoglobin.

Additionally, the solution can contain a variety of additionalcomponents, including, but not limited to, antibacterial and antifungalagents and red blood cell lysing agents.

The solution is maintained in a sealed environment under anaerobicconditions until shortly before use. When its use as a control isdesired, the solution can be equilibrated with physiological gasses viaa method such as tonometry. The level of methemoglobin remains low dueto the presence of the thio groups and metal ion catalyst which act tocatalytically reduce any methemoglobin which might form duringtonometry. Once equilibrated, the system provides values of pH, pCO₂ andpO₂ suitable for monitoring Blood Gas/pH analysis instrumentation. Thesolution also provides physiological levels of hemoglobin and fractionsof hemoglobin species for calibrating and monitoring CO-Oximetryinstrumentation. The solution can also be used as a reference solutionfor electrolytes (Na⁺, K⁺, Ca⁺⁺, Cl⁻). Finally the solution alsoexhibits oxygen saturation characteristics similar to those of freshwhole blood.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a stable reference solution for, inter alia,blood gas and CO-Oximetry instrumentation. Unlike solutions previouslyemployed for calibration and monitoring of blood gas/CO-Oximetryequipment in which physiological gasses are present in solution duringstorage, the present solution comprises a stable, aqueous, hemoglobinsolution which is equilibrated with physiological gasses just prior touse. This solution, and equilibration method associated therewith,provide a blood gas reference solution having a storage life which islonger than that of previously employed controls. This is achieved inpart through the use of a metal ion catalyzed methemoglobin reducingsystem which serves to inhibit formation of undesirable methemoglobinduring storage, gas equilibration and use.

A cell-free hemoglobin solution is prepared from healthy animal blood.In the preferred embodiment, the hemoglobin source is bovine, ratherthan human, blood. Although modified human red blood cell solutions, aswell as stroma-free human hemoglobin solutions, are commerciallyavailable, these human-source solutions are susceptible to contaminationby a variety of human infectious agents including the hepatitis virusand HIV. The use of non-human blood products in blood-based referencesolutions is therefore desirable. As such, the use of bovine bloodcollected from healthy animals is preferred due to its similarity tohuman blood in both physical and chemical characteristics.

Additionally, the present invention exhibits oxygen saturationcharacteristics which are more similar to those of fresh whole bloodthan have been seen in other control standards. For example, when oxygensaturation is plotted against partial pressure, known referencesolutions exhibit a hyperbolic curve. In contrast, the present solution,like fresh whole blood, exhibits a sigmoid curve. Furthermore, a valuefor the required oxygen partial pressure to achieve 50% saturation, theso-called P₅₀ value, has been determined for the reference solution ofthis invention. This P₅₀ values, obtained for tonometered solutions madefrom bovine erythrocyte derivatives, were in the range of 18 to 34 mmHg,with a nominal value of 26. This value is very close to that obtainedfrom fresh human blood, generally about 27 mmHg.

The solution of red blood cells is repeatedly washed with saline.Subsequently, the washed red blood cells are combined with deionizedwater. At this point, some red blood cell lysis may occur. Next, theremaining red blood cells are lysed by the addition of a lysing agent.In the preferred embodiment, the lysing agent is benzethonium chloride.This compound is selected because it also provides antibacterial andantifungal activity to the solution in addition to acting as a celllysing agent. Optionally at this point, an additional compound, such asthe preferred 2-phenoxyethanol, can be added to the solution to provideadditional antibacterial and antifungal activity to the solution. Theresulting solution contains completely lysed red blood stroma andhemoglobin in solution. The stroma can be removed at this point bycentrifugation or it can be removed after subsequent processing steps.

The methemoglobin reducing system is now added to the completely lysedhemoglobin solution. The preferred methemoglobin reducing systemcomprises a sulfhydro compound selected from the class of chemicalswhich have an effective thio (--SH) functional group. Such chemicalsinclude, but are not limited to the reduced form of glutathione,mercaptoethanol, cysteine and their derivatives. A catalytic amount of ametal ion is provided to the solunion via salts of selenium, tellurium,and copper which are added to the solution to catalyze the methemoglobinreduction reaction. Such a metal ion catalyzed methemoglobin reducingsystem is described by Masukawa and Iwata in Life Sciences, 21, (5), 695(1977), the teachings of which are incorporated herein by reference. Inthis reference, selenite, selenate, and selenocystine are described ascatalysts for reactions in which methemoglobin is reduced byglutathione, 2-mercaptoethylamine and cysteine.

Additionally, further resistance to methemoglobin formation can beimparted to the solution by the addition of a polyphosphate compound.This compound stabilizes the hemoglobin molecules in a spatialconfiguration which is not readily oxidized to the methemoglobin form.Preferred polyphosphate compounds include 2,3-diphosphoglycerate,inositol hexaphosphate (also referred to as phytic acid), and adenosinetriphosphate. The use of a polyphosphate to stabilize hemoglobin wasdescribed in greater detail by Mansouri in Hemoglobin, 5(6), 579 (1981),the teachings of which are incorporated herein by reference.

Following the addition of the hemoglobin stabilizing systems describedabove, the pH of the solution is adjusted to within a physiologicalrange. In the preferred embodiment, the pH is adjusted to between about7.0 and 7.6 using aqueous NaOH under an inert atmosphere such asnitrogen or noble gas. A source of bicarbonate ion, preferably sodiumbicarbonate and a suitable organic buffer selected from Good's buffersand their derivatives are added to buffer the pH. Preferable buffersinclude N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES;pK_(a) =7.31), 3-[N-bis(hydroxyethyl) -amino]-2-hydroxypropane sulfonicacid (DIPSO; pK_(a) =7.35), piperazine-N-N'-Bis (2-hydroxypropanesulfonic acid) (POPSO; pK_(a) =7.63),3-[N-(Tris-hydroxymethyl)methylamino ]-2-hydroxypropane sulfonic acid(TAPSO; pK_(a) =7.39), and N-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO; pK_(a) =7.73).

The solution is passed through a series of membrane filters having afinal filter of a maximum pore size of 0.2 um to remove any red bloodcell stroma which remains. Optionally, an antifoaming agent can be addedat this point.

The hemoglobin solution is then further bubbled with nitrogen or otherinert gasses which contain between about 4 to about 7% carbon dioxide.This nitrogen bubbling step serves to convert any oxyhemoglobin insolution to reduced hemoglobin and to maintain anaerobic conditions. Thecarbon dioxide in the gas mixture is used to maintain an acceptable pHvalue between about 7.0 and 7.2 in the solution. The solution is thensealed in a container, such as glass ampule, under an atmosphere likethat bubbled through the solution.

The resulting hemoglobin contained in the aqueous solution preferablycomprises hemoglobin fractions of at least about 95% reduced hemoglobin,less than 3% methemoglobin, less than 2% oxyhemoglobin, less than 2%carboxyhemoglobin and less than 0.3% by volume oxygen. The solution hasa purple-blue color as a result of the reduced hemoglobin containedtherein. As there are few, if any, oxidizing agents (including molecularoxygen) in solution, the formation of methemoglobin is almost completelyeliminated. As there is only negligible methemoglobin in solution, themethemoglobin reducing system is not utilized at this point. Rather, thesystem will serve to prevent methemoglobin production during asubsequent gas equilibration just prior to solution use.

When the solution is to be used for monitoring of blood gas/CO-Oximetryinstruments, it must first be equilibrated with a physiological gasmixture. In the preferred method, this equilibration is performed viatonometry. Tonometry is the term used to describe the process whereby aliquid is brought into equilibrium with a gas phase by bubbling thedesired gas through the liquid or by forming a liquid film with largesurface area interacting with the incoming gas. A detailed discussion oftonometry is provided by R. W. Burnett in Clincal Chemistry, 27(10),1761 (1981), the teachings of which are incorporated herein byreference. During the tonometry (or even when exposed to ambient air)the solution rapidly changes from the purple-blue color indicative ofthe presence of reduced hemoglobin to a red color indicative of thepresence of oxyhemoglobin. The amount of methemoglobin produced duringthe tonometry remains minimal due to the metal ion catalyzedmethemoglobin reduction system described previously.

Upon completion of the equilibration, the solution will contain known,predetermined levels of pH, pCO₂ and pO₂. Thus the solution can beanalyzed by a Blood Gas/pH Analyzer and can be used to monitor theinstrumentation and ensure a minimum drift from the set-point. Thesolution can also be used for the calibration and monitoring ofCO-Oximetry equipment used to measure levels of total hemoglobin andfractions of hemoglobin species.

In a preferred embodiment, the solution is representative of normalhuman blood. In this embodiment, the solution has the followingcomposition:

    ______________________________________                                        Component        Range       Specific                                         ______________________________________                                        Total Bovine     13-15 g/dl  14 g/dl                                          Hemoglobin                                                                    2-Phenoxyethanol 0.09-0.11 g/dl                                                                            0.1 g/dl                                         Benzethonium Chloride                                                                          0.09-0.11 g/dl                                                                            0.1 g/dl                                         Sodium Selenite  1-20 μM  2 μM                                          Phytic Acid      0.1-1.4 mM  0.14 mM                                          Reduced Glutathione                                                                            0.5-8 mM    0.8 mM                                           Sodium Bicarbonate                                                                             28.5-30.5 mM                                                                              29.16 mM                                         HEPES            20-40 mM    25 mM.                                           ______________________________________                                    

Upon use, this solution is equilibrated via tonometry with a gas mixturewhich comprises by volume 5% CO₂, 12% O₂ and 83% N₂. This equilibrationis carried out at 37° C. until the pH is within the range of about 7.38to about 7.42, the pCO₂ is within the range of about 34 to about 38mmHg, and the pO₂ is within the range of about 83 to about 89 mmHg. In amost preferred embodiment, following equilibration, the solution willhave a pH of about 7.4, a pCO₂ of about 36 mmHg and a pO₂ of about 86mmHg at 37° C.

In another preferred embodiment, the solution is representative of theblood of a human patient experiencing acidosis. In this embodiment, thesolution has the following composition:

    ______________________________________                                        Component         Range        Specific                                       ______________________________________                                        Total Bovine      8-10 g/dl    9 g/dl                                         Hemoglobin                                                                    2-Phenoxyethanol  0.09-0.11 g/dl                                                                             0.1 g/dl                                       Benzethonium Chloride                                                                           0.09-0.11 g/dl                                                                             0.1 g/dl                                       Sodium Selenite   1-20 μM   2 μM                                        Phytic Acid       0.1-1.4 mM   0.14 mM                                        Reduced Glutathione                                                                             0.5-8 mM     0.8 mM                                         Sodium Bicarbonate                                                                              19.5-21.5 mM 20.5 mM                                        HEPES             20-40 mM     25 mM.                                         ______________________________________                                    

Upon use, this solution is equilibrated via. tonometry with a gasmixture which comprises by volume 7% CO₂, 7% O₂ and 86% N₂. Thisequilibration is carried out at 37° C. until the pH is within the rangeof about 7.12 to about 7.18, the pCO₂ is within the range of about 48 toabout 52 mmHg, and the pO₂ is within the range of about 47 to about 53mmHg. In a most preferred embodiment, following equilibration, thesolution will have a pH of about 7.15, a pCO₂ of about 50 mmHg and a pO₂of about 50 mmHg at 37° C.

In yet another preferred embodiment, the solution is representative ofthe blood of a human patient experiencing alkalosis. In this embodiment,the solution has the following composition:

    ______________________________________                                        Component        Range       Specific                                         ______________________________________                                        Total Bovine     17-19 g/dl  18 g/dl                                          Hemoglobin                                                                    2-Phenoxyethanol 0.09-0.11 g/dl                                                                            0.1 g/dl                                         Benzethonium Chloride                                                                          0.09-0.11 g/dl                                                                            0.1 g/dl                                         Sodium Selenite  1-20 μM  2 μM                                          Phytic Acid      0.1-1.4 mM  0.14 mM                                          Reduced Glutathione                                                                            0.5-8 mM    0.8 mM                                           Sodium Bicarbonate                                                                             19-21 mM    19.82 mM                                         HEPPSO           20-40 mM    25 mM                                            ______________________________________                                    

Upon use, this solution is equilibrated via tonometry with a gas mixturewhich comprises by volume 2.8 CO₂, 20% O₂ and 77.2% N₂. Thisequilibration is carried out at 37° C. until the pH is within the rangeof about 7.58 to about 7.62, the pCO₂ is within the range of about 19 toabout 21 mmHg, and the pO₂ is within the range of about 140 to about 144mmHg. In a most preferred embodiment, the solution will have a pH ofabout 7.6, a pCO₂ of about 20 mmHg and a pO₂ of about 142 mmHg at 37° C.

A preferred batch protocol for producing a large volume of referencesolution representative of normal blood is given below:

Reagents:

(A) Bovine red blood cell suspension

(B) Aqueous solution containing 2% each of 2-Phenoxyethanol andBenzethonium Chloride

(C) Aqueous solution containing 0.072% Na₂ SeO₃ and 1.87% Phytic Acid

(D) 119.15 g HEPES plus 11.03 g NaOH in 1 liter of deionized water toprovide a net pH of about 7.4

(E) 4.92 g reduced glutathione in 1 liter deionized water

Procedures:

1) Wash the bovine red blood cell suspension with saline solution atleast four times.

2) Adjust the total hemoglobin content to 18.0 g % using deionizedwater, (some red cell lysis may occur at this point).

3) Complete the red cell lysis by adding 50 ml of reagent (B) to 800 mlof the partially lysed red cell suspension, adding a small amount at atime and mixing thoroughly to avoid hemoglobin aggregation.

4) Add 50 ml each of reagents (C) and (D) to 850 ml of the completelylysed hemoglobin solution obtained in step (3).

5) Add 50 ml of reagent (E) to bring the total volume to one liter.

6) Adjust the pH of the solution to 7.4 by slowly adding a 4% aqueousNaOH solution under a nitrogen (100%) atmosphere. (The total volume ofNaOH added should be recorded to determine whether addition of reagentspreviously described is necessary.)

7) Add 2.45 g/l solid NaHCO₃ to the solution.

8) Filter the hemoglobin solution through a filter having a maximum poresize of about 0.2 um.

9) Add a small quantity of a silicone-based defoamer such as antifoam Aconcentrate to the solution.

10) Flush the hemoglobin solution with nitrogen gas containing about 4to 7% CO₂ at room temperature until the oxyhemoglobin value is less than1% of the total hemoglobin.

11) Subdivide the solution into individual sealed ampules having the gasmixture of step 10) contained in the head space.

The resulting, sealed ampules of reference solution can be stored atroom temperature for a period of at least about three months and at 4°C. for at least about one year. When used for monitoring ofblood-gas/CO-Oximetry equipment is desired, the ampule is opened andsubjected to tonometry with a predetermined gas mixture as previouslydescribed. The resulting reference solution has many characteristics offresh, human whole blood including physiological ranges of pH, pCO₂,pO₂, total hemoglobin content and fractions of hemoglobin species.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be covered by the following claims.

What is claimed is:
 1. A stable hemoglobin reference solution comprisingan aqueous solution containing:a) hemoglobin; b) a source of bicarbonateions; c) a source of thio functional groups; d) a source of metal ioncatalyst; e) at least one organic polyphosphate; and, an organic buffer.2. A solution of claim 1 wherein the hemoglobin comprises 95% reducedhemoglobin.
 3. A stable reference solution of in claim 2 having a P₅₀value of between about 18 to about 34 mmHg after tonometry with anappropriate gas mixture of O₂, CO₂ and N₂.
 4. A stable referencesolution of claim 3 wherein the hemoglobin comprises mammalianhemoglobin.
 5. A stable reference solution of claim 4 wherein thehemoglobin comprises bovine hemoglobin.
 6. A stable reference solutionof claim 5 wherein thio functional groups are provided by a sulfhydrocompound selected from reduced glutathione, mercaptoethanol, cysteine orderivatives thereof.
 7. A stable reference solution of claim 6 whereinthe source of metal ion catalyst comprises a salt of metals selectedfrom the group consisting of selenium, tellurium and copper.
 8. A stablereference solution of claim 7 wherein the organic polyphosphate compoundis selected from the group consisting of 2,3-diphosphoglycerate, phyticacid and adenosine triphosphate.
 9. A stable reference solution of claim1 contained in a sealed container having a head space containing CO₂ anda gas which is inert to the reference solution.